Diseases caused by insect-borne kinetoplastid parasites are marked by their prevalence in poverty-stricken populations and their lack of safe and effective treatment strategies. An understanding of the unusual processes of gene regulation in kinetoplastids, best studied in Trypanosoma brucei, may uncover superior drug targets for these pathogens. Evidence suggests that turnover of the 18 mitochondrial mRNAs plays a significant role in gene regulation. Attempts to identify and describe the regulation of cis elements recognized by exoribonucleases have met with limited success in T. brucei mitochondria, partly because mitochondrial homologues of known exoribonucleases do not comprise the primary RNA turnover machinery. Non-encoded 3' nucleotide tails impact RNA stability in all cellular compartments, and the 3' tails observed in trypanosome mitochondria may be cis-acting stability factors as well. Limited data obtained to date suggest that 3' tails of T. brucei mitochondrial mRNAs are strikingly varied among transcripts but are consistent in composition and length for a particular transcript. Our hypothesis is that the length, nucleotide composition, and/or the complexity of 3' non-encoded RNA tails comprise a stability code that is read by 3'-5' exoribonucleases. Furthermore, these 3' extensions may be the reason for the varied mitochondrial transcript abundances observed between the insect and mammalian life cycle stages of the parasite. In Aim 1, we will for the first time directly determine relative stabilities of a selected group of mitochondrial transcripts within and between life cycle stages by attenuating transcription and measuring RNA decay rates over time utilizing qRT-PCR. Aim 2 undertakes the fundamental characterization of 3' non-encoded tails of the same mitochondrial RNAs by sequencing 20-40 circular RT-PCR products for each transcript, again in two life cycle stages. Tail characteristics will be compared with the stabilities obtained in Aim 1 in paired and regression analyses to determine specifically the relationship between 3' tails and transcript stability within and between life cycle stages. In parallel with the above approaches, our final aim is to identify the primary mitochondrial exoribonuclease responsible for a robust decay activity observed in mitochondrial protein extracts by purifying this protein through sequential chromatography steps, determining its identity by LC-MS/MS, and showing its impact on RNA turnover in vivo by examining mitochondrial RNA abundance in cells depleted for this enzyme. The long-term goal of this project is to understand the role of RNA stability within the larger scope of post-transcriptional regulation in trypanosome mitochondrial gene expression. In summary, the research proposed here will provide insight into the mechanisms of mitochondrial RNA turnover in the medically and economically relevant kinetoplastid parasites. These studies may uncover unique aspects of kinetoplastid biology that can be exploited in the future for development of new chemotherapeutics.